Toll-like receptors (TLRs) mediate specific recognition of pathogens and inflammatory signaling in macrophages during the host's innate immune response. Mycobacterium tuberculosis (MTh) and Leishmania species are intramacrophage pathogens which cause significant morbidity and mortality worldwide and serve as model pathogens for examining the immune response. While many microbes, including MTh, stimulate macrophages through a TLRdependent pathway, the nature and extent of TLR influence on the innate and adaptive immune response is only beginning to be elucidated. The hypothesis of this proposal is that TLRs are pivotal molecules in vivo during the immune response to MTh and Leishmania infections and that they mediate inflammatory signaling pathways that influence both pathogen and host survival. The aims of this proposal are to examine the effects of TLR2 in MTh and Leishmania major mouse infection models and to characterize TLR-dependent inflammatory signaling pathways in vitro in macrophages infected with L. major. The in vivo function of TLR2 will be assessed by constructing transgenic mice with macrophage- specific expression of a dominant-negative TLR2 mutation (TLR2DNmac). This novel cell-specific dominant-negative strategy offers several advantages over a knockout approach and will be a powerful tool for exploring the in vivo significance of these molecules. TLR2DNmac, TLR2KO (knockout), and TLR4KO mice will be infected with aerosolized MTh or subcutaneously with L. major and then examined for pathogen load, inflammatory cell recruitment to the infection site, and the induction of T cell and macrophage effector responses to determine which aspects of the host response are influenced by TLRs. In addition to their medical importance, Leishmania species are unusual in their capacity to live in the harsh environment of the macrophage where they exert anti-inflammatory effects as a survival strategy. While the signaling mechanisms that enable Leishmania to exist in the macrophage are largely unknown, preliminary evidence presented in this proposal suggests that TLR pathways are involved. TLR-mediated cytokine production will be characterized in vitro in L. major-infected macrophages to determine the molecular identity of these signaling pathways. The cooperation of TLRs in L. major recognition will also be examined to elucidate how macrophages specifically respond to different microbial ligands. Advances in the understanding of the role of TLRs in macrophages during the immune response to M. tuberculosis and L. major will reveal novel insights about innate immunity and infectious diseases that will have implications for vaccine development.